Post-exposure therapy of influenza  a infections

ABSTRACT

Poly ICLC or liposome-encapsulated Poly ICLC (LE Poly ICLC) in combination with antisense oligonucleotides (AS) act synergistically in post-exposure prophylaxis or therapy of influenza infections, especially H5N1 virus infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 61/438,704 filedFeb. 2, 2011, the entire contents of which is herein incorporated byreference. Also, the Sequence Listing filed electronically herewith ishereby incorporated by reference (File name:2012-02-02T_(—)1036-0001_Seq List; File size: 2 KB; Date recorded: Feb.2, 2012).

FIELD OF THE INVENTION

The present invention relates to compositions and methods forpost-exposure treatment of viral infections, particularly influenza Avirus infections.

BACKGROUND OF THE INVENTION

Influenza viruses are etiological agents of deadly flu that continue topose global health threats and have caused global pandemics that killedmillions of people worldwide. The global crisis posed by the emergenceof the avian H5N1 influenza virus provides testament to the challengesof defending against a deadly virus that is unpredictable and everchanging. As the human death toll from the bird flu outbreaks continuesto increase, the world is moving closer to a potential influenzapandemic. With a reported case fatality over 50%, an influenza pandemicby a highly transmissible strain of avian H5N1 influenza virus couldpotentially kill millions of people worldwide.

There are a number of commercially available antiviral drugs which canbe considered as the first line of defense against seasonal or avianinfluenza infections. There are two conventional influenza drugs, namelyamantadine and rimantadine, which have been used clinically to reducethe severity and duration of seasonal flu. These compounds arc M2ion-channel blocking adamantine derivatives that work by interferingwith viral uncoating inside the cells. In recent years, good advanceshave been made in the development of anti-influenza drugs, particularlythe development of neuramidase inhibitors, namely Tamiflu™ (Zanamivir)[4-acetamido-5-amino-3-(1-ethylpropoxyl)-1-cyclohexene-1-carboxylic acidethyl ester (invented by Gilead Sciences and marketed by Roche AG, andRelenza™ (Oseltamivir) (4-guanidino-Neu5Ac2en) (marketed byGlaxo-SmithKline). Neuraminidase is one of the glycoproteins present onthe virus surface, and it is an enzyme that cleaves sialic acid residuesfrom the receptors for the virus. The inhibition of this enzyme byTamiflu™ and Relenza™ stops the spread of this virus and effectivelysuppresses infection.

One of the greatest challenges with current stockpiling of antiviraldrugs as part of pandemic planning or bioterrorism defense is high levelof drug-resistance of influenza viruses. Most seasonal human influenzastrains (H3N2) are resistant to amantadine and rimantadine, therebylimiting their usefulness against seasonal influenza. When thedistribution of genetic mutations associated with resistance of avianH5N1 influenza virus to amantadine and rimantadine was determined, itwas found that more than 95% of isolates of avian H5N1 influenza fromThailand and Vietnam contained resistance mutations to these drugs.While many western nations are stockpiling drugs such as Tamiflu™ aspart of the pandemic preparedness plan, there is a growing concern thatthe circulating avian flu virus may be resistant to Tamiflu™.Significant number of the avian flu clinical isolates from hard-hitareas in Vietnam had developed resistance to Tamiflu™. As global fearsof a pandemic grow, the use of neuraminidase inhibitors against seasonalinfluenza has been shown to significantly increase, and this will leadto corresponding incidence of drug-resistance to these drugs. Anotherchallenge posed by stockpiling of Tamiflu™ is that for the drug to beeffective, it has to be given within 48 hours after exposure. Once thesymptoms of infection appear the therapeutic efficacy of the drugsdecreases dramatically. To better defend against influenza, whetherseasonal and/or pandemic, there are compelling reasons to develop noveldrugs or drug combinations that are safe, effective and less likely togive rise to drug-resistance.

Poly ICLC is a synthetic, double-stranded RNA (ds RNA) comprised ofpolyriboinosinic-polyribocytidylic (I:C) acid stabilized withpoly-L-lysine and carboxymethylcellulose. Poly ICLC is a potentimmunomodulating agent (Wong 2007). Liposome encapsulated Poly ICLC (LEPoly ICLC) was found to be a broadly effective prophylactic against anumber of seasonal and avian influenza viruses in pre-exposure therapies(Wong 2007). LE Poly ICLC was also found to provide high level ofprotection in mice against highly pathogenic H5N1 avian influenza Avirus. The window of protection provided by LE Poly ICLC was determinedby pre-treating mice with 2 intranasal (IN) doses (1 mg/kg/dose) of LEPoly ICLC at day 1 to day 20 prior to infection with 10 LD₅₀ ofinfluenza A/PR/8/34 virus. Although LE Poly ICLC when given toexperimental animals in a pre-exposure prophylactic mode has been shownto provide complete protection against a number of influenza virusstrains, including H5N1, H3N2 and H1N1 subtypes, its efficacy whenadministered in a post-exposure treatment mode after viral infectionremains to be determined.

Ribonucleotide oligonucleotides (RNOs) are known to provide antiviralprotection, including protection against influenza A viruses. Liposomeencapsulated RNOs have been shown to be effective in the post-exposuretreatment of mice infected with influenza A virus (Wong 2003). Otherantisense oligonucleotides useful against influenza virus infections areknow (Mizuta 1999; Zhang 2010).

There is a need in the art for new therapies against viral infections,in particular against influenza A virus.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that Poly ICLC orliposome-encapsulated Poly ICLC (LE Poly ICLC) in combination withantisense oligonucleotides (AS) act synergistically in post-exposureprophylaxis or therapy of influenza infections.

Thus, there is provided a method of treating an influenza virusinfection in a subject comprising administering to the subject asynergistic combination of Poly ICLC or liposome-encapsulated Poly ICLC(LE Poly ICLC) and one or more antisense oligonucleotides (AS) after thesubject has been exposed to the influenza virus.

There is further provided a use of a synergistic combination of PolyICLC or liposome-encapsulated Poly ICLC (LE Poly ICLC) and one or moreantisense oligonucleotides (AS) for treating an influenza virusinfection in a subject after the subject has been exposed to theinfluenza virus.

There is further provided a pharmaceutical composition for post-exposuretreatment of an influenza virus infection in a subject, the compositioncomprising a synergistic mixture of Poly ICLC or liposome-encapsulatedPoly ICLC (LE Poly ICLC) and one or more antisense oligonucleotides(AS).

There is further provided a use of LE Poly ICLC for treating aninfluenza virus infection in a subject after the subject has beenexposed to the influenza virus.

Poly ICLC is a large double-stranded RNA (ds RNA) that is greater than200 nucleotides in length. It is a broad-spectrum drug what worksagainst a wide range of influenza virus isolates. The mechanisms bywhich Poly ICLC elicits broad antiviral immune responses are mediatedthrough recognition by and interaction with toll-like receptor-3 (TLR-3)on dendritic cells, monocytes and macrophages of the host's innateimmunity (Wong 2009b). This recognition of ds RNA ligand by TLR-3induces the production of interferon-α, interferon-β and interferon-γ invivo. This signaling pathway also plays a significant role forstimulation of both innate and adaptive immune responses, including theactivation of natural killer cells.

Antisense oligonucleotides (AS) are short single-stranded highlyvirus-specific RNA/DNA oligomers. AS are preferably 13-25 nucleotideslong, for example, about 15 nucleotides long. Antisense oligonucleolidesmediate inhibiting of influenza A virus replication by a gene silencingapproach. Haemagglutinin (HA) plays an important and essential role inthe pathogenesis of influenza viral infection. Antisenseoligonucleotides are capable of binding to the haemagglutinin (HA) gene,thereby inhibiting HA gene transcription, and/or are capable of bindingto the transcribed HA mRNA, thereby inhibiting HA protein synthesis,and/or are capable of binding to the HA protein of the influenza virusparticle itself, thereby inhibiting the action of HA protein, andeffectively inhibiting viral replication and proliferation.

A variety of suitable antisense oligonucleotides are known in the art.For example, U.S. Pat. No. 6,544,958 discloses several suitableribonucleotide oligonucleotides (RNOs). Antisense oligonucleotides maybe unencapsulated or may be encapsulated in liposomes. Antisenseoligonucleotides may comprise one or more modified bases, for example2′-O-methyl RNA modified bases. Antisense oligonucleotides may compriseone or more non-nucleotide-based spacers, for example C1, C2, C3, C4 orC5 spacers. Preferred antisense oligonucleotides comprise SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, chemically modified variants thereof (e.g.SEQ ID NO: 4) or mixtures thereof.

In addition to synergy, the present combination treatment offers adistinct advantage over conventional anti-influenza treatment withantiviral drugs in that the present combination treatment is more robustand less likely to give rise to drug resistance. Poly ICLC, including LEPoly ICLC, and antisense oligonucleotides work independently withdistinct mechanisms of action as discussed above. It is thereforeunlikely that influenza A viruses can simultaneously mutate to evadeboth of these mechanisms, especially since TLR-3 on the host's immunedefense cell are not knows to mutate and change. Thus, due to its broadspectrum effect, Poly ICLC and LE Poly ICLC are well suited to providean effective and robust antiviral agent that complements antisenseoligonucleotides to combat seasonal, zoonotic and pandemic influenzaviruses. Since Poly ICLC and LE Poly ICLC do not exert antiviralactivity by specifically targeting influenza virus structure or protein,it is less likely to give rise to drug-resistance and may thereforeoffer protection against various influenza viruses, regardless ofgenetic mutations, reassortments, recombinations, zoonotic origin ordrug-resistance.

Poly ICLC or LE Poly ICLC is typically used in an amount effective totreat the influenza virus infection. In combination with the one or moreantisense oligonucleotides, the amount of Poly ICLC or LE Poly ICLC usedis sufficient to act synergistically with the one or more antisenseoligonucleotides. A suitable dosage of Poly ICLC or LE Poly ICLC may bewithin a range of from about 0.1 mg/kg body weight to about 5 mg/kg bodyweight, or about 0.5 mg/kg body weight to about 2 mg/kg body weight, forexample about 1 mg/kg body weight. Antisense oligonucleotides aretypically used in an amount effective to treat the influenza virusinfection and to act synergistically with the Poly ICLC or LE Poly ICLC.A suitable dosage of AS may be within a range of from about 1 mg/kg bodyweight to about 50 mg/kg body weight, or about 5 mg/kg body weight toabout 35 mg/kg body weight, for example about 20 mg/kg body weight.

Dosing regimes for a particular subject may be determined by a treatingphysician. Ideally, a first dose is administered to the subject as soonas possible after the subject has been exposed to the virus. However, itis a great advantage of the present combination therapy that treatmentcan be initiated well after initial exposure, for example 24 hourspost-infection or even 48 hours post-infection, with the subject stillhaving excellent survival prospects. Subjects may be dosed any suitablenumber of times per day, preferably 1-3 times per day, for example 1time per day. Subjects may be further dosed in subsequent days.Preferably, subjects are dosed every day after the first dose for up toabout 3 days.

The Poly ICLC or LE Poly ICLC and antisense oligonucleotides may beadministered to a subject simultaneously or serially within a shortperiod of time of each other. Preferably, Poly ICLC or LE Poly ICLC andthe antisense oligonucleotides are part of the same pharmaceuticalcomposition, but they can be administered separately provided they areadministered within a time sufficient that the synergistic action is notlost. Typically, the time for clearing one of the compounds from thebody is an extreme upper bound on the time to administer the other.However, administration of the other within 6 hours, more preferably 2hours, even more preferably 1 hour, yet more preferably 30 minutes, ofthe first is preferred.

Any suitable administration method known in the art may be used. Someadministration methods include intranasal, intravenous, intraarterial,subcutaneous or enteric. Intranasal administration is preferred.Preferred dosage forms for intranasal administration include aerosols,nasal sprays and nasal instillations. Active ingredients may beadministered as part of a pharmaceutical composition “as is” or togetherwith one or more pharmaceutically acceptable carriers, diluents orexcipients. Active ingredients may form part of a kit or commercialpackage together with instructions for use in treating influenza virusinfections.

The present post-exposure therapy is effective for treating seasonaland/or pandemic influenza infections. The therapy is useful againsthighly pathogenic avian influenza viruses. The therapy is particularlyuseful against known and emerging influenza A infections. Some examplesof known influenza A infections are H1N1, H3N2, H5N1 and H5N2infections. It is especially effective against H5N1 (e.g. HPA1) virusinfections, which are often difficult to treat. The combination of LEPoly ICLC and antisense oligonucleotides is particularly effective.

Subjects that may be treated include animals, for example, mammals,birds (chickens, turkeys, pea fowl, etc.) and reptiles. Mammaliansubjects include, for example, humans, horses, cows, sheep, goats, pigs,dogs, cats, rabbits, mice, hamsters, guinea pigs, monkeys, chimpanzeesand rats.

Further features of the invention will be described or will becomeapparent in the course of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, embodimentsthereof will now be described in detail by way of example, withreference to the accompanying drawings, in which:

FIG. 1 depicts graphs showing therapeutic effect of post-exposuretreatment with LE Poly ICLC alone (1 mg/kg body weight) on mice infectedintranasally with 5 LD₅₀ H5N1 virus. Post-exposure treatments were doneat 30 min post-infection or 24 hours post-infection, followed bytreatment at same dose once per day for three days. In FIG. 1A, survivalof six mice in each group was observed for 14 days post-infection anddata are expressed as percentages of total survival. For FIG. 1B, threemice in each group were euthanized on day 4 post-infection and viraltiters in lung were determined by plaque assay and Real-time PCR (*,p<0.001).

FIG. 2 depicts a graph showing therapeutic effect of post-exposurecombination treatment of LE Poly ICLC (1 mg/kg body weight) andantisense oligonucleotide (SEQ ID NO: 4, 20 mg/kg body weight) on miceinfected intranasally with 5 LD₅₀ H5N1 virus. Post-exposure treatmentswere done at 24 hours post-infection, followed by treatment at same doseonce per day for three days. Survival of six mice in each group wasobserved for 14 days post-infection and data are expressed aspercentages of total survival.

FIG. 3 depicts a graph showing synergistic effect of Poly ICLC andantisense oligonucleotide (SEQ ID NO: 4) for the post exposure treatmentof influenza A/PR/8/34 virus in mice.

FIG. 4 depicts a graph showing synergistic effect of low dose of LE PolyICLC and antisense oligonucleotide (SEQ ID NO: 4) for the post exposuretreatment of influenza A/PR/8/34 virus in mice.

FIG. 5 depicts a graph showing synergistic effect of high dose of LEPoly ICLC and antisense oligonucleotide (SEQ ID NO: 4) for the postexposure treatment of influenza A/PR/8/34 virus in mice.

DESCRIPTION OF PREFERRED EMBODIMENTS

Materials and Methods:

Female BALB/c mice (8-10 weeks old) were obtained from Vital RiverLaboratories (Beijing, China), and the original breeding pairs werepurchased from Charles River. Mice were raised in IVC (independentventilated cages) and received pathogen-free food and water.

Antisense oligonucleotides (AS) were prepared by Oligos Etc Inc.(Wilsonsville, Oregon, USA). For all experiments, all AS were diluted in0.9% sodium chloride made from RNAse-free water (Sigma, St. Louis, Mo.).The sequence of the AS used was as follows:

(SEQ ID NO: 4) 5′-mUmCmGmUmUmUmUmCmGmUmCmCmCmCmUX-3′where m represents a 2′-O-methyl RNA modified base, and X is a C4 spacer(based on butanol).

Phospholipids and cholesterol used for the preparation ofliposome-encapsulated Poly ICLC were purchased from Avanti Polar Lipids(Alabaster, Ala.). Large multilamellar vesicles (MLVs) were preparedusing phosphatidylcholine, cholesterol and phosphatidyl glycerol in amolar ratio of 7:2:1, and were prepared using freeze drying procedurespreviously described (Wong 2007).

Example 1 Post-Exposure Treatment of Mice Exposed to Influenza A Virus

To evaluate the efficacy of LE Poly ICLC and antisense oligonucleotidecombination treatment against highly pathogenic avian influenza (H5N1)viral infection, groups of 8 to 10 BALB/c mice were randomly assigned aseither negative control animal group (saline only), LE Poly ICLC onlyanimal group or LE Poly ICLC and AS animal group. The groups were thenchallenged by intranasally infecting them with 5 LD₅₀ ofinfluenza/H5N1/Henan/chicken (a representative HPA1) virus.

At 30 minutes post virus challenge, a negative control group was treatedintranasally with phosphate buffered saline, an LE Poly ICLC alone groupwas treated intranasally with LE Poly ICLC (1 mg/kg body weight/dose)and an LE Poly ICLC and AS group was treated with LE Poly ICLC (1 mg/kgbody weight/dose) and AS (20 mg/kg body weight/dose), followed byintranasal administration once per day for three days at the same dose.At 24 hours post virus challenge, another negative control group,another LE Poly ICLC alone group and another LE Poly ICLC and AS groupwere dosed using the same dosing regime as above. The volume of theinoculum in all cases was 50 μl. The animals were then monitored dailyfor symptoms of infection, body weight and survival. At day 14 postinfection, the number of mice which survived the virus infection in eachgroup was recorded. Survival patterns of the mice were graphed using theLog-rank test (GraphPad Prism version 4.01, San Diego, Calif.).Differences were considered significant at p<0.05.

Although previously published results have demonstrated that LE PolyICLC has a prophylactic effect on controlling H5N1 viral replication invivo when administered to subjects prior to viral exposure (Wong 2007),there has been no previous report on its therapeutic efficacy inpost-exposure treatment. In the present example, the LE Poly ICLC alonegroup provides results for the effect of LE Poly ICLC in post-exposuretreatment. The results (FIG. 1A) show that 50% of the mice that weretreated with LE Poly ICLC 30 min post infection survived to 14 days postinfection, while the mice treated 24-hour post infection all died by 10days post infection. All of the mice in the negative control group diedby 9 days post infection. This reflects the importance of treatmenttiming when using LE Poly ICLC alone as a post-exposure therapeuticagent. Pulmonary viral titer, as measured in by plaques in MDCK cells inlung tissues of the animals, showed a similar trend to survival rate(FIG. 1B).

Therapeutic efficacy of a combination treatment 24 hours post-infectionusing LE Poly ICLC and antisense oligonucleotides is shown in FIG. 2 incomparison to the negative control group (saline only) and the LE PolyICLC alone group. The results showed that treatment with LE Poly ICLCalone at 24 hours post-infection was only partially effective resultingin 50% survival rate. However, when combined with antisenseoligonucleotide (SEQ ID NO: 4), all of the H5N1 infected animalssurvived the virus challenge (100% survival, p<0.001 vs. negativecontrol). That all treated animals given a combination of LE Poly ICLCand antisense oligonucleotide were completely protected against anunnaturally high virus challenge evidences the synergism of thiscombination in treating H5N1 virus infection.

Example 2 Synergistic Effect of Poly ICLC or LE Poly ICLC Together withAntisense Oligonucleotides Against Influenza A/PR/8/34 (H1N1) VirusInfection

Therapeutic efficacies of combination treatment using free Poly ICLC orLE Poly ICLC and antisense oligonucleotides (AS) were further evaluatedto confirm that free Poly ICLC or LE Poly ICLC act synergistically withantisense oligonucleotides to treat influenza A/PR/8/34 virus infectionin mice.

Mice were challenged with 5 LD₅₀ influenza A/PR/8/34 (H1N1) virus, andthen intranasally given free Poly ICLC (1.2 mg/kg), LE Poly ICLC (1.2mg/kg), antisense oligonucleotide (SEQ ID NO: 4, 15 mg/kg), acombination of free Poly ICLC and antisense oligonucleotide, or acombination of LE Poly ICLC and antisense oligonucleotide at 1 and 4hours post infection.

Post exposure treatment of influenza A infection using free Poly ICLCalone (1.2 mg/kg) was not effective and did not result in any increasedsurvival while antisense oligonucleotide provided a 50% survival rate(FIG. 3). However, when infected mice were treated with a combination offree Poly ICLC and SEQ ID NO: 4, all treated mice survived the virusinfection while all PBS control treated mice died from infection (FIG.3) (p<0.01). The 100% survival rate with combination treatment washigher than the combined survival rates from single treatments with PolyICLC and SEQ ID NO: 4, indicating synergistic therapeutic effectobtained with combination therapy with Poly ICLC and SEQ ID NO: 4.

Similarly, antisense treatment at a lower dose of SEQ ID NO: 4 (150μg/mouse) was not effective (0% survival), and neither was LE Poly ICLC(20 μg/mouse, 10% survival), but combined therapy using these doses ofLE Poly ICLC and SEQ ID NO: 4 provided a synergistic increased survivalrate of 40% (FIG. 4).

Similar results were observed at the same dose of LE Poly ICLC (20μg/mouse) and higher doses of SEQ ID NO: 4 (250 μg/mouse) given singlyor in combination therapy (FIG. 5). Single treatment with SEQ ID NO: 4(0% survival) and LE Poly ICLC (10%) was not very effective, whilecombination therapy with LE Poly ICLC and SEQ ID NO: 4 resulted insignificantly high synergistic therapeutic efficacy of more than 70%(FIG. 5).

Free Listing of Sequences:

SEQ ID Sequence SEQ ID NO: 1 5′ UCC CCU GCU UUU GCU 3′(15 nucleotides, from U.S. Pat. No. 6,544,958) SEQ ID NO: 2 5′AGC AAA AGC AGG GGA 3′ (15 nucleotides, from U.S. Pat. No. 6,544,958)SEQ ID NO: 3 5′ UCG UUU UCG UCC CCU 3′ (15 nucleotides, fromU.S. Pat. No. 6,544,958) SEQ ID NO: 4 5′ mUmCmG mUmUmU mUmCmGmUmCmC mCmCmU X 3′ (15 nucleotides, 2′-O-methyl modified, X is a C4 spacer)

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Other advantages that are inherent to the structure are obvious to oneskilled in the art. The embodiments are described herein illustrativelyand are not meant to limit the scope of the invention as claimed.Variations of the foregoing embodiments will be evident to a person ofordinary skill and are intended by the inventor to be encompassed by thefollowing claims.

1. A method of treating an influenza virus infection in a subjectcomprising administering to the subject a synergistic combination ofPoly ICLC or liposome-encapsulated Poly ICLC (LE Poly ICLC) and one ormore antisense oligonucleotides (AS) after the subject has been exposedto the influenza virus.
 2. The method of claim 1, wherein thesynergistic combination comprises LE Poly ICLC and one or more antisenseoligonucleotides.
 3. The method of claim 2, wherein the influenza virusis an influenza A virus.
 4. The method of claim 2, wherein the influenzavirus is H1N1, H3N2, H5N1 or H5N2 virus.
 5. The method of claim 2,wherein the influenza virus is H5N1 virus.
 6. The method of claim 2,wherein the one or more antisense oligonucleotides comprises anucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ IDNO:
 3. 7. The method of claim 2, wherein the one or more antisenseoligonucleotides comprises a nucleotide sequence as set forth in SEQ IDNO:
 4. 8. The method of claim 2, wherein the one or more antisenseoligonucleotides are administered in liposome-encapsulated form.
 9. Themethod of claim 2, wherein the LE Poly ICLC is administered in an amountin a range of from 0.1 to 5 mg/kg body weight of the subject.
 10. Themethod of claim 2, wherein the one or more antisense oligonucleotides isadministered in an amount in a range of from 1 to 50 mg/kg body weightof the subject.
 11. The method of claim 2, wherein the LE Poly ICLC andthe one or more antisense oligonucleotides are administered to thesubject within 2 hours of each other.
 12. The method of claim 2, whereinthe LE Poly ICLC and the one or more antisense oligonucleotides areadministered to the subject simultaneously.
 13. The method of claim 2,wherein the LE Poly ICLC and the one or more antisense oligonucleotidesare administered intranasally.
 14. The method of claim 13, whereinintransal administration is accomplished with an aerosol.
 15. The methodof claim 2, wherein the subject is human.